Three OsMYB36 members redundantly regulate Casparian strip formation at the root endodermis.

Plants have evolved a lignin-based Casparian strip (CS) in roots that restricts passive diffusion of mineral elements from the soil to the stele. However, the molecular mechanisms underlying CS formation in rice (Oryza sativa), which contains a CS at both the exodermis and endodermis, are poorly understood. Here, we demonstrate that CS formation at the rice endodermis is redundantly regulated by three MYELOBLASTOSIS (MYB) transcription factors, OsMYB36a, OsMYB36b, and OsMYB36c, that are highly expressed in root tips. Knockout of all three genes resulted in a complete absence of CS at the endodermis and retarded plant growth in hydroponic conditions and in soil. Compared with the wild-type, the triple mutants showed higher calcium (Ca) levels and lower Mn, Fe, Zn, Cu, and Cd levels in shoots. High Ca supply further inhibited mutant growth and increased Ca levels in shoots. Transcriptome analysis identified 1,093 downstream genes regulated by OsMYB36a/b/c, including the key CS formation gene OsCASP1 and other genes that function in CS formation at the endodermis. Three OsMYB36s regulate OsCASP1 and OsESB1 expression by directly binding to MYB-binding motifs in their promoters. Our findings thus provide important insights into the mechanism of CS formation at the endodermis and the selective uptake of mineral elements in roots.

Overexpression of OsCASP1 Improves Calcium Tolerance in Rice.

The Casparian strip domain protein 1 (OsCASP1) is necessary for the formation of the Casparian strip (CS) in the rice endodermis. It also controls Ca2+ transport to the stele. Here, we demonstrated that OsCASP1 overexpression enhanced Ca tolerance in rice. Under normal conditions, OsCASP1-overexpressed lines showed similar concentrations of essential metals in the roots and shoots compared to the wild type, while under high Ca conditions, Ca in the roots, shoots, and xylem sap of the OsCASP1-overexpressed lines was significantly decreased. This did not apply to other essential metals. Ca-inhibited growth was significantly alleviated in the OsCASP1-overexpressed lines. Furthermore, OsCASP1 overexpression resulted in earlier formation of both the CS and functional apoplastic barrier in the endodermis but did not induce ectopic CS formation in non-endodermal cell layers and affect suberin accumulation in the endodermis. These results indicate that the overexpression of OsCASP1 promotes CS formation in endodermal cells and inhibits Ca2+ transport by the apoplastic pathway, restricting Ca accumulation in the roots and shoots under high Ca conditions. Taken together, the results suggest that OsCASP1 overexpression is an effective way to improve rice adaptation to high Ca environments.

Disruption of an amino acid transporter LHT1 leads to growth inhibition and low yields in rice.

BACKGROUND: Research on plant amino acid transporters was mainly performed in Arabidopsis, while our understanding of them is generally scant in rice. OsLHT1 (Lysine/Histidine transporter) has been previously reported as a histidine transporter in yeast, but its substrate profile and function in planta are unclear. The aims of this study are to analyze the substrate selectivity of OsLHT1 and influence of its disruption on rice growth and fecundity. RESULTS: Substrate selectivity of OsLHT1 was analyzed in Xenopus oocytes using the two-electrode voltage clamp technique. The results showed that OsLHT1 could transport a broad spectrum of amino acids, including basic, neutral and acidic amino acids, and exhibited a preference for neutral and acidic amino acids. Two oslht1 mutants were generated using CRISPR/Cas9 genome-editing technology, and the loss-of-function of OsLHT1 inhibited rice root and shoot growth, thereby markedly reducing grain yields. QRT-PCR analysis indicated that OsLHT1 was expressed in various rice organs, including root, stem, flag leaf, flag leaf sheath and young panicle. Transient expression in rice protoplast suggested OsLHT1 was localized to the plasma membrane, which is consistent with its function as an amino acid transporter. CONCLUSIONS: Our results indicated that OsLHT1 is an amino acid transporter with wide substrate specificity and with preference for neutral and acidic amino acids, and disruption of OsLHT1 function markedly inhibited rice growth and fecundity.

METTL14/IGF2BP2-mediated m6A modification of STEAP1 aggravates acute lung injury induced by sepsis.

BACKGROUND: Acute lung injury (ALI) is a severe complication of sepsis, characterized by inflammation, edema, and injury to alveolar cells, leading to high mortality rates. Septic ALI is a complex disease involving multiple factors and signaling pathways. STEAP family member 1 (STEAP1) has been reported to be upregulated in a sepsis-induced ALI model. However, the role of STEAP1 in the regulation of septic ALI is not yet fully understood. METHODS: The study stimulated human pulmonary microvascular endothelial cells (HPMECs) using lipopolysaccharides (LPS) to establish an in vitro ALI model. The study used quantitative real-time polymerase chain reaction (qRT-PCR) to measure mRNA expression, and western blotting assay or immunohistochemistry (IHC) assay to analyze protein expression. Cell counting kit-8 (CCK-8) assay was performed to assess cell viability. Flow cytometry was conducted to analyze cell apoptosis. Tube formation assay was used to analyze the tube formation rate of human umbilical vein endothelial cells (HUVECs). Enzyme-linked immunosorbent assays (ELISAs) were used to measure the levels of interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α). The levels of Fe2+ and reactive oxygen species (ROS) were determined using colorimetric and fluorometric assays, respectively. The glutathione (GSH) level was also determined using a colorimetric assay. m6A RNA immunoprecipitation assay, dual-luciferase reporter assay, and RNA immunoprecipitation assay were performed to identify the association of STEAP1 with methyltransferase 14, N6-adenosine-methyltransferase non-catalytic subunit (METTL14) and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2). The transcript half-life of STEAP1 was analyzed by actinomycin D assay. Finally, a rat model of polymicrobial sepsis was established to analyze the effects of STEAP1 knockdown on lung injury in vivo. RESULTS: We found that the mRNA expression levels of STEAP1 and METTL14 were upregulated in the blood of ALI patients induced by sepsis compared to healthy volunteers. LPS treatment increased the protein levels of STEAP1 and METTL14 in HPMECs. STEAP1 depletion attenuated LPS-induced promoting effects on HPMECs' apoptosis, inflammatory response, and ferroptosis, as well as LPS-induced inhibitory effect on tube formation. We also found that METTL14 and IGF2BP2 stabilized STEAP1 mRNA expression through the m6A methylation modification process. Moreover, METTL14 silencing attenuated LPS-induced effects by decreasing STEAP1 expression in HPMECs, and STEAP1 silencing ameliorated cecal ligation and puncture-induced lung injury of mice. CONCLUSION: METTL14/IGF2BP2-mediated m6A modification of STEAP1 aggravated ALI induced by sepsis. These findings suggest potential therapeutic targets for the treatment of this disease.

The tonoplast-localized transporter OsABCC9 is involved in cadmium tolerance and accumulation in rice.

Cadmium (Cd) is a highly toxic element to living organisms, and its accumulation in the edible portions of crops poses a potential threat for human health. The molecular mechanisms underlying Cd detoxification and accumulation are not fully understood in plants. In this study, the involvement of a C-type ABC transporter, OsABCC9, in Cd tolerance and accumulation in rice was investigated. The expression of OsABCC9 was rapidly induced by Cd treatment in a concentration-dependent manner in the root. The transporter, localized on the tonoplast, was mainly expressed in the root stele under Cd stress. OsABCC9 knockout mutants were more sensitive to Cd and accumulated more Cd in both the root and shoot compared to the wild-type. Moreover, the Cd concentrations in the xylem sap and grain were also significantly increased in the knockout lines, suggesting that more Cd was distributed from root to shoot and grain in the mutants. Heterologous expression of OsABCC9 in yeast enhanced Cd tolerance along with an increase of intracellular Cd content. Taken together, these results indicated that OsABCC9 mediates Cd tolerance and accumulation through sequestration of Cd into the root vacuoles in rice.

Amino Acid Transporters in Plant Cells: A Brief Review.

Amino acids are not only a nitrogen source that can be directly absorbed by plants, but also the major transport form of organic nitrogen in plants. A large number of amino acid transporters have been identified in different plant species. Despite belonging to different families, these amino acid transporters usually exhibit some general features, such as broad expression pattern and substrate selectivity. This review mainly focuses on transporters involved in amino acid uptake, phloem loading and unloading, xylem-phloem transfer, import into seed and intracellular transport in plants. We summarize the other physiological roles mediated by amino acid transporters, including development regulation, abiotic stress tolerance and defense response. Finally, we discuss the potential applications of amino acid transporters for crop genetic improvement.

OsCASP1 forms complexes with itself and OsCASP2 in rice.

OsCASP1 (Casparian strip domain protein 1) was recently identified to function in Casparian strip (CS) formation at the endodermal cells in rice roots, which was required for selective mineral uptake in rice. Here, we further investigate the functional form of OsCASP1 in vivo. Expression analysis shows that OsCASP1, OsCASP2, OsCASP3, and OsCASP5 were expressed in roots apart from OsCASP4. A yeast two-hybrid (Y2H) assay revealed that OsCASP1 can interact with itself and OsCASP2, but not with OsCASP3 and OsCASP5. These interactions of OsCASP1 with itself and OsCASP2 at the plasma membrane were confirmed using bimolecular fluorescence complementation (BiFC) in rice protoplasts. These results indicated that OsCASP1 can form complexes with itself and OsCASP2 in rice roots.